Transactivation of EGF receptor induced by G protein-coupled receptor stimulation : the study for signaling cascade by Angiotensin II

血管紧张素II诱导的大鼠血管平滑肌细胞和心脏成纤维细胞中EGF受体的反式激活：使用免疫沉淀和蛋白质印迹分析，发现通过1型AngII受体（AT 1）的AngII刺激可使EGF-R磷酸化。这种磷酸化可被Ca^2+螯合剂、Ca^2+/钙调蛋白抑制剂和酪氨酸激酶抑制剂完全消除。此外，特异性抑制EGF-R的显性负性EGF-R突变体和tyrphostin AG 1478废除了激活细胞外信号调节激酶（ERK）的AT 1，导致抑制纤连蛋白（FN）基因表达的AT 1。利用FN-CAT融合基因的启动子分析表明，AP-1结合位点负责FN基因的AT 1反应性。G1期阻滞实验表明，AG 1478预处理可抑制AT 1刺激的c-jun和c-fos与FN基因AP-1位点的结合。总之，血管紧张素II诱导的EGF-R的反式激活是由Ca^2+/钙调蛋白依赖性酪氨酸激酶介导的。ERK的激活和随后的基因表达，如FN由AT 1通过EGF-R反式激活的下游信号调节。

Transactivation of EGF-receptor induced by AngiotensinII in rat vascular smooth muscle cells and cardiac fibroblasts : Using immunoprecipitation and Western blot analysis, AngII stimulation via type 1 AngII receptor (AT1) was found to phosphorylate EGF-R This phosphorylation was completely abolished by the pretreatment of Ca^<2+> chelating agents, Ca^<2+>/calmodulin inhibitors and tyrosine kinase inhibitors. In addition, specific inhibition of EGF-R by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished the activation of extracellular signal-regulated kinase (ERK) by AT1, leading to the suppression of fibronectin (FN) gene expression by AT1. The promoter analysis using FN-CAT fusion gene revealed that AP-1 binding site was responsible for the AT1 responsiveness of FN gene. G1 retardation assay showed that the pretreatment of AG1478 inhibited AT1-stimulated binding of c-jun and c-fos to AP-1 site in FN gene. In summary, the transactivation of EGF-R induced by angiotensin II is mediated by Ca^<2+>/calmodulin-dependent tyrosine kinases. The activation of ERK and subsequent gene expression such as FN by AT1 were regulated through downstream signaling of EGF-R transactivation.